Quorum sensing is a process of cell-to-cell communication that bacteria use to assess their population density in order to coordinate the gene expression of the community (Miller et al., 2001). Quorum sensing requires the production, secretion, and detection of extra-cellular signal molecules termed autoinducers. Diverse behaviors are controlled by quorum sensing, but, typically, these behaviors are ones that would be ineffective if only a small group of cells carried them out. Often, bacteria produce and detect multiple autoinducers, some of which are used for intraspecies communication, while others promote interspecies communication (Federle et al., 2003; Fuqua et al., 2001; Xavier et al., 2003).
The marine bacterium Vibrio harveyi produces and detects two autoinducers, AI-1 and AI-2, and these signals control the expression of multiple genes, including those for bioluminescence (luciferase) (Bassler et al., 1993, 1994a; Cao et al., 1989; Chen et al., 2002b), siderophore production (Lilley et al., 2000), colony morphology, metalloprotease production (Mok et al., 2003), and type III secretion (Henke et al., 2004).
In V. harveyi, AI-1 and AI-2 are produced by the synthases LuxM and LuxS, respectively (Bassler et al., 1993; Surette et al., 1999). LuxN detects AI-1, and LuxPQ detects AI-2 (FIG. 1A) (Bassler et al., 1993, 1994a; Chen et al., 2002b; Freeman et al., 2000b). LuxN and LuxQ are membrane bound, two-component hybrid sensor-kinase proteins. LuxP, which binds AI-2 in the periplasm, is required with LuxQ for the response to AI-2 (Bassler et al., 1994a; Chen et al., 2002b). Sensory information from both systems converges at the phosphorelay protein LuxU, and LuxU transmits the signal to the response regulator LuxO (Bassler et al., 1994b; Freeman et al., 1999, 2000a). A transcriptional activator called LuxR is also required for expression of lux (the operon encoding the bioluminescence genes in V. harveyi) and other quorum sensing-controlled genes (Henke et al., 2004; Martin et al., 1989; Miyamoto et al., 1994; Showalter et al., 1990).
The human pathogen Vibrio cholerae possesses quorum-sensing systems analogous to the two described above for V. harveyi (Miller et al., 2002). The V. cholerae autoinducers CAI-1 and AI-2 are synthesized by CqsA and LuxS and detected by CqsS and LuxPQ, respectively (FIG. 1B). V. cholerae has an additional system (System 3; Miller et al., 2002). Sensory information from all three systems converges at LuxO. The V. cholerae LuxR homolog is called HapR (Jobling et al., 1997). Quorum sensing controls virulence and biofilm formation in V. cholerae (Hammer et al., 2003; Kovacikova et al., 2002; Miller et al., 2002; Vance et al., 2003; Zhu et al., 2003; Zhu et al., 2002).
The V. harveyi and V. cholerae quorum-sensing circuits operate similarly (Miller et al., 2002). At low cell density, i.e., in the absence of autoinducers, the sensors act as kinases and transfer phosphate via LuxU to LuxO. LuxO-phosphate (LuxO-P) is active and negatively regulates lux. At high cell density, i.e., when the autoinducers are present, the sensors act as phosphatases. Phosphate flow through the circuit is reversed, resulting in dephosphorylation and inactivation of LuxO (Freeman et al., 1999, 2000a; Freeman et al., 2000b). Under this condition, the transcriptional regulators LuxR in V. harveyi and HapR in V. cholerae bind the lux promoter and activate transcription (FIG. 1).
LuxO-P-mediated repression of lux is indirect (Lilley et al., 2000). LuxO is homologous to members of the NtrC family of response regulators, which can act either as transcriptional activators or repressors. Those that are activators require the alternative sigma factor σ54 for function, while those that are repressors do not (Benson et al., 1994; North et al., 1996; Reitzer et al., 1985; Wingrove et al., 1994; Wu et al., 1997). LuxO is a member of the activator class of NtrC homologs. It has been hypothesized that, at low cell density, LuxO-P activates the expression of a repressor that controls the downstream target genes.
The present invention relates to the discovery that multiple, redundant small regulatory RNAs (sRNAs), together with the sRNA binding protein Hfq, fulfill this repressor role. Specifically, at low cell density, the Hfq-sRNA repressor complexes destabilize the V. harveyi luxR and V. cholerae hapR mRNAs (FIG. 1).